The presence of pathogenic cells in body fluids, such as the bloodstream, or the spread of pathogenic cells from other sites to the bloodstream is one of the important factors that determines whether or not a diseased patient will survive. For example, the spread of malignant cells from the primary neoplasm to distant organs is an important factor in determining whether cancer patients will survive. Highly sensitive methods must be developed that can detect and/or quantify circulating pathogenic cells, such as metastatic cells, and other types of pathogenic cells in the vasculature at the earliest stages of disease. Achievement of this objective requires highly sensitive, biocompatible probes with selectivity for the pathogenic cells.
Currently, CT, MRI, tissue/sentinel lymph node biopsy, and serum cancer marker analysis can detect some level of residual disease in cancer patients; however, the presence of tumor cells in circulation (CTCs) correlates most sensitively with cancer prognosis and metastasis. Additionally, flow cytometry is a method that is used for detecting and quantitating target cell populations using fluorescent probes that render high specificity and low background measurements. Flow cytometry assays can be performed in vivo without acquisition of a patient sample or ex vivo using a sample of a patient body fluid acquired from the patient. However, prior, conventional ex vivo techniques have many deficiencies. For example, the methods typically require the use of a large sample due to low sensitivity of the methods.
More recently, a conceptual in vivo flow cytometry technique utilizing confocal microscopy has been described for the real-time detection of flowing tumor cells in vivo (Georgakoudi, et al. (2004) Cancer Res., 64, 5044-5047; Novak, et al. (2004) Optics Lett., 29, 77-79). However, there is no available method with the requisite sensitivity and biocompatibility for performing the detection and quantification of pathogenic cells in an ex vivo sample of a patient body fluid. In this application, Applicants describe the detection and quantification of pathogenic cells found in patient body fluids using an ex vivo sample of a patient body fluid and using flow cytometry-based techniques.
In one embodiment, a method is described for diagnosing a disease state mediated by pathogenic cells. The method comprises the steps of combining with an ex vivo patient sample a composition comprising a conjugate or complex of the general formula Ab-X, wherein the group Ab comprises a ligand that binds to the pathogenic cells and the group X comprises an imaging agent, and detecting the pathogenic cells that express a receptor for the ligand using flow cytometry.
In another embodiment, a method is described for determining a prognosis of a cancer by detecting cancer cells in an ex vivo patient sample. The method comprises the steps of detecting the cancer cells in the ex vivo patient sample by flow cytometry, and determining a prognosis for the cancer.
In another embodiment, a method is described for quantitating pathogenic cells. The method comprises the steps of combining with an ex vivo patient sample a composition comprising a conjugate or complex of the general formula Ab-X, wherein the group Ab comprises a ligand that binds to the pathogenic cells and the group X comprises an imaging agent, and quantitating the pathogenic cells in the ex vivo patient sample using flow cytometry.